Our broad, long-term objective is to determine why mutations of the murine agouti locus like lethal yellow (Ay) and viable yellow (Avy) cause pathologies of the Lethal Yellow Syndrome (LYS)-obesity, aberrant pigmentation, cancer, enhanced growth, immunosuppression, infertility, and diabetes. The agouti locus regulates the type of pigment (black and/or yellow) synthesized within follicular melanocytes (Silvers, 19790. Agouti alleles alter specific melanogenic parameters (Ito, 1993; Tamate et al., 1989; Granholm et al., 1990b). With the advent of agouti gene isolation, cloning, sequencing, and partial characterization (Bultman et al., 1992; Miller et al., 1993), we now for the first time have an opportunity to correlate the molecular events of melanogenesis with virtually unequivocal evidence of agouti expression (i.e., presence of agouti transcripts followed by synthesis of agouti proteins). Specific Aims include: 1) Developmental Northern analyses together with specific assays of melanogenesis (tyrosinase (both tyrosine hydroxylase and dopa oxidase), type of melanin (total, eu-, and phaeomelanin), dopachrome tautomerse (DT), and melanosome differentiation via transmission electron microscopy) on regenerating and newborn hair bulbs of Strain C57BL\6J wild-type agouti(AwJ/AwJ), lethal yellow (Ay/a), and nonagouti black (a/a) genotypes and 2) Northern and Western analyses of extrafollicular "target tissues" of the lYS (fat stores-obesity, ovarian follicles-infertility, pituicytes- potential MSH lesions, and splenocytes-immunosuppression); we plan to determine the chronology of agouti expression and assess whether Ay-induced pathologies in these tissues arise autonomously or by systemic action of agouti proteins made elsewhere. With respect to human health, experiments designed to correlate agouti expression with precise components of melanogenesis will provide refined information on how the agouti locus, one of about 50 genetic loci involved in the regulation of pigmentation, determines the balance of black, yellow, or both pigments within follicular melanocytes. An understanding of the agouti-encoded regulation of this "pigment equilibrium" (i.e., switch from black-to-yellow-to-black) should be productive for the diagnosis and treatment of melanomas an other pigment cell disorders, because we will possess a more fundamental knowledge of pigment cell metabolism and therefore a greater potential for drug intervention. Studies on aberrant LYS "target tissues" of adult Ay/a mice may allow us to identify molecular lesions common to those same conditions in humans -obesity, cancer, abnormal growth, compromised immunity, infertility, and diabetes resulting potentially in the development of new therapies. Because agouti expression apparently depends upon a signaling mechanism, we may discover that the common lesion uniting many of the pathologies of the LYS is a fundamental defect in cell signaling. Elucidation of the molecular bases of an Av- induced signaling lesion may yield important data on similar signaling lesions in humans.